1. Field of the Invention
The present invention relates to an anti-pathogen system that kills or injures pathogen-infected cells by introducing into the cells a fusion protein comprising a protein transduction domain and a cytotoxic domain. The cytotoxic domain is capable of being specifically activated in cells infected by the pathogen. Further provided are specified transduction domains that enhance transduction capacity of the fusion protein. The present invention has a variety of uses such as killing or injuring cells infected by one or more pathogenic viruses or plasmodia.
2. Background
A variety of pathogens infect mammals, particularly primates such as humans. For example, certain viruses, bacteria, fungi, yeasts, worms, plasmodia, and protozoa are recognized human pathogens. See e.g., Harrison's Principles of Internal Medicine, 12.sup.th ed. McGraw-Hill, Inc. (1991).
Pathogens often kill or injure cells by mechanisms that manifest morphological characteristics. For example, pathogen-infected cells undergoing apoptosis or necrosis extLibit readily identifiable cellular changes.
There has been progress toward understanding cell proteins and particularly enzymes, involved in apoptosis. For example, certain cell proteases such as caspases (i.e. cysteinyl aspartate-specific proteases), C. elegans ced-3 and granzyme B have been implicated in apoptosi. Nucleic acid sequences encoding several capsases and proteolytic substrates for same are known. For example, caspase-3 (i.e. CPP32) has been particularly well-studied. See e.g., Thompson, C. B. Science, 267:1456 (1995); and Walker, N. P. C. et al. Cell, 78:343 (1994).
There have been related attempts to identify proteins involved in necrosis. For example, necrosis is thought to follow expression of certain DNA viruses such as herpes viruses.
Pathogens often induce synthesis of certain proteins, particularly enzymes such as proteases. It is likely that nearly all pathogens require one or more specific proteases to complete a productive infection. For example, it is believed that the following exemplary human pathogens require expression of at least one pathogen-specific protease: cytomegalovirus (CMV), herpes simplex virus, e.g., type-1 (HSV-1); hepatitis virus, e.g., tyrpe C (HCV); certain plasmodia, e.g., P. falciparum; human immunodeficiency virus type 1 (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV); human immunodeficiency virus type 2 (HIV-2), Kaposi's sarcoma-associated herpes virus (KSHV or human herpes virus 8), yellow fever virus, certain flaviviruses and rhinovirus.
Sometimes the proteases are encoded by the pathogen itself. In this instance, the proteases are often referred to as pathogen-specific proteases. For example, CMV, HCV, HIV-1, HIV-2, KSHV, and P. falciparum are representative of pathogens that encode pathogen-specific proteases. These proteases serve a variety of functions and can be nearly indispensable for a productive infection.
There has been some efforts to analyze particular pathogen specific proteases such as serine-type proteinases encoded by HCV, aspartic proteases (i.e. plasmepsins I and II) encoded by P. falciparum, and a maturational protease encoded by HSV-1. See e.g., Dilanni, C. L. et al., J. Biol. Chem., 268:2048 (1993); and Francis, S. E. et al., EMBOJ., 13:306 (1994).
In contrast, inducible expression of certain host cell proteases is believed to modulate productive infection by other pathogens. These host cell proteases are sometimes referred to as inducible host cell proteases. For example, bacterial infection of eukaryotes such as certain plants can induce expression of normally quiescent host cell proteases. Induction of the host cell proteases may be an attempt to damage the pathogen, thereby protecting the host cell from infection.
Infection by HIV viruses has attracted substantial attention. There is now almost universal agreement that the human family of these retroviruses are the etiological agent of acquired immune deficiency syndrome (AIDS) and related disorders. Productive infection by nearly all HIV viruses requires expression of certain HIV-specific proteases. See, for example, Barre-Sinoussi et al., Science, 220:868-871 (1983); Gallo et al., Science, 224:500-503 (1984).
There has been progress toward developing therapeutic agents to target pathogen infections such as HIV infections. One general approach has focussed on interrupting distinct stages of the pathogen infection. In particular, therapeutic agents have been developed to combat certain HIV specific enzymes such as reverse transcriptase (RT) and pathogen-specific proteases.
Other agents such as certain cytokines have been used in attempts to treat CMV and HSV infections.
Other proposed methods for treating pathogen infections relate to what has been referred to as "intracellular immunization". Briefly, the methods involve genetically modifying host cells in an attempt to render them incapable of supporting a productive infection. For example, it has been suggested that certain eukaryotic cells can be made immune to pathogen infection by using the method. See e.g., Baltimore, Nature, 335:739:5 (1988); Harrison et al., Human Gene Therapy, 3:461 (1992); and U.S. Pat. No. 5,554,528 to Harrison et al.
A more specific form of genetic modification has been reported to involve administering gene constucts that encode cytotoxins. In this instance, the contructs are desigened so that the genes can express cytoxin once inside the cells.
However, the prior methods for treating pathogen infections have several limitations.
For example, methods that use a cytotoxin to kill cells have not always been successful. One explanation may relate to pleiotropic effects reported for many intracellular cytotoxins. Those effects can often complicate analysis of cell killing. Additionally, many gene constructs that encode a cytotoxin can exhibit undesirably high basal activities inside host cells. These problems can produce what is known as "leaky" cytotoxin expression, leading to death of infected and non-infected cells.
Other methods for treating pathogen infection have also had problems. For example, methods relying on use of a drug have not been completely effective. More particularly, subjects infected by aggressive or persistent pathogens often require prolonged therapeutic intervention, sometimes over a period of months or even years. Proliferation of drug resistant pathogens is becoming increasingly problematic. Thus, the long-term value of the methods is controversial.
In particular, current treatment of HIV utilizes small inhibitory molecules that target HIV protease. However, emergence of resistant HIV strains is increasingly problematic. See e.g., Coffin, J. M., et al. Retroviruses, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1997); Kaplan, A. H. et al. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease. Proc. Natl. Acad. Sci. USA 91: 5597 (1994); Condra, J. H. et al. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 374: 569 (1995); Gulnik, S. V. et al. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry 34: 9282 (1995); and Tisdale, M. et al. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors, Antimicrob. Agents Chemother. 39:1704 (1995).
There has been recognition that the retroviral TAT protein may find use in certain therapuetic settings. TAT has been reported to transactivate certain HIV genes and it is believed to be essential for productive infection by most human HIV retroviruses. The TAT protein has been used to bring certain types of fusion proteins into cells. This process is generally referred to as transduction. See U.S. Pat. No. 5,652,122 to Frankel et al.; and Chen, L. L. et al., Anal. Biochem., 227:168 (1995).
However, use of TAT to transduce fusion proteins into cells has been associated with significant shortcomings.
For example, it has been difficult to maintain suitable levels of the fusion proteins inside cells. Attempts to overcome this problem have included administering large amounts of the fusion proteins to help maintain adequate intracellular levels. The need to administer large amounts of the fusion proteins may prevent or hinder widespread use of some therapeutic fusion proteins. For example, use of large amounts of some TAT fusion proteins may negatively impact viability of some host cells.
Further, it has been difficult to maintain many of the prior transducing fusion proteins in a therapeutically relevant conformation. As an illustration, it is believed that many prior TAT fusion proteins may partially or completely unfold during transduction. That unfolding has potential to significantly reduce or eliminate transduction in many instances.
Additionally, the need to correctly fold the prior transducing fusion proteins has complicated efforts to purify and store the proteins.
Further drawbacks have been reported to be associated with proteins fused to TAT or certain TAT fragments. These drawbacks relate to how TAT is believed to act inside cells. More specifically, there has been acknowledgement that TAT or the TAT fragments may confer certain biological characteristics to the fusion proteins. Some of these characteristics and particularly nuclear localization and RNA binding may not always be desirable. In particular, there has been concern that many TAT fusion proteins may be difficult to position outside the nucleus or away from RNA. See e.g., Dang et al., J. Biol. Chem., 264:18109 (1989); Calnan, B. J. et al., Genes Dev., 5:201 (1991) for a discussion of TAT-associated properties.
It would be desirable to have an anti-pathogen system that exhibits high transduction efficiency and can specifically deliver a cytotoxin to pathogen infected cells. It would be further desirable to have an anti-pathogen system that can deliver the cytotoxin as an essentially inactive molecule that can be activated by pathogen infected cells.